(a) Field of the Invention
The invention relates to a novel DNA assay for the diagnosis and/or prediction of autoimmune diabetes.
(b) Description of Prior Art
Diabetes is a major cause of morbidity and mortality in industrialized societies. It has been estimated that one of every seven health-care dollars goes to treating diabetes and its complications. Type 1 diabetes (also called insulin-dependent or juvenile diabetes, henceforth referred to in this document as xe2x80x9cdiabetesxe2x80x9d) is due to the autoimmune destruction of the insulin-producing pancreatic xcex2-cells. Type 1 diabetes is less common than type 2, accounting for only 10-20% of cases in Caucasians. However, because it is much more severe and starts much earlier in life, it accounts for a large proportion of diabetes-related morbidity and mortality.
Type 1 diabetes involves autoimmune destruction of the insulin-producing pancreatic xcex2-cells. Insulin, an autoantigen in this process, is expressed in human thymus at levels dependent on alleles at the upstream INS VNTR, to which the IDDM2 susceptibility locus has been mapped. Chromosomes carrying the dominantly protective (Bennett S T et al., 1995, Nat. Genet. 9(3):284-292), long INS VNTR alleles (class III) produce 2-3 times higher levels of insulin gene (INS) mRNA than those with predisposing, short class I alleles (Vafiadis P et al., 1997, Nat Genet. 15(3):289-292; Pugliese A et al., 1997, Nat. Genet. 15(3):293-297).
It is estimated that by the time symptoms of diabetes appear, more than 95% of the xcex2-cell mass has been destroyed. Given the irreversibility of this destruction, the most promising approach to the disease is prevention. This will require an intervention, at some time before symptoms appear, aimed at modulating the immune system to prevent the antigen-specific autoimmune reaction. Although the specific causative autoantigen(s) in diabetes is (are) not known, insulin, the main product of the xcex2-cell, appears to be an autoantigen of major importance.
Based on this, the DPT (diabetes prevention trial) a large study, is now underway in the United States. Oral insulin administration is used in the hope of helping individuals at risk for diabetes acquire immune tolerance to insulin. The results of this study will not be known for a number of years. It is possible that it will be superseded by other studies, which will be based on more precise scientific rationale provided by current spectacular advances in immunology. There is a tangible likelihood that in the coming decade a safe and effective method of preventing or reversing the diabetes autoimmune process will have been found.
An effective intervention to prevent diabetes is very unlikely to be inexpensive, safe and convenient enough to be applied to the general population (i.e. in the fashion of infectious disease vaccines). The intervention will most likely need to be targeted to individuals that can be identified as being at a substantial risk for diabetes.
The DPT and similar trials have focused on first-degree relatives of diabetics that are positive for autoantibodies known to predict diabetes. These autoantibodies against protein components of the xcex2-cell become positive at least a year or two before the onset of clinical diabetes. However, even if diabetes is prevented with 100% efficacy in all first-degree relatives of diabetics, this will only abolish less than 10% of new cases of diabetes, as the majority of individuals with diabetes do not have a previously affected first-degree relative. To be meaningful, an effective prevention will have to be applicable to the general population. Screening the general population for antibodies has been shown to be feasible in practice, and predictive of diabetes. However, since antibodies only become positive at a finite time point before the onset of diabetes, most people destined to become diabetic at some future point will be negative when tested early in life. Therefore, antibody screening of the general population will need to be repeated at intervals of 1-2 years, a totally impractical proposition.
An alternative way of predicting high risk for diabetes is through DNA testing. It is known that genetic predisposition plays a major role in diabetes. Identical twins of individuals with diabetes have a risk that ranges from 30 to  greater than 60% in various studies, as opposed to fraternal twins, that are concordant only in 5-10% of the cases (Bennett S T et al., 1996, Ann. Rev. Genet. 30:343-370). The predisposition is not inherited in a Mendelian fashion, which means that this complex phenotype requires predisposing genetic material involving more than a single gene. Linkage studies have identified as many as 18 different genetic loci that are potentially linked to diabetes (in genetics, the term xe2x80x9clocusxe2x80x9d is used instead of xe2x80x9cgenexe2x80x9d, when only a location in the genome is known, but the gene[s] involved remain[s] to be identified). Although many of these loci will probably turn out to be statistical artifacts, it is clear that in order to define the xe2x80x9cdiabetes genotypexe2x80x9d, markers at more than one loci will have to be typed. A good definition of the xe2x80x9cdiabetes genotypexe2x80x9d would be: a combination of alleles that correctly predicts diabetes with a probability that approaches that predicted from being the identical twin of a diabetic. Obviously, a risk of at least 30% would justify preventive intervention.
So far only two of the several loci have been defined precisely enough to be used to this end. They are termed IDDM1 and IDDM2, and can be used already to obtain a certain degree of risk estimation (Bennett S T et al., 1996, Ann. Rev. Genet. 30:343-370). Using similar approaches several more loci can be likewise defined, that together predict diabetes with specificity that approaches that of the idealized diabetes genotype. Specificity in a diagnostic test is the percentage of positive tests that turn out to be true. The twin studies show that 30-60% is the best specificity that can be achieved with DNA testing, but even a specificity of 10% or less can be very useful depending on how safe, simple and inexpensive future preventive interventions turn out to be.
Once defined, the loci could be genotyped on a few drops of blood obtained at birth as part of neonatal screening programs currently in place for other diseases. Those individuals who exceed a certain threshold of risk can then be followed with antibody testing or treated prior to the appearance of autoantibodies, depending on what the optimal strategy will be determined to be.
It involves determination of the VNTR by Southern blotting, a cumbersome technique that is not easily amenable to miniaturization and automation (Bennett S T et al., 1995, Nat. Genet. 9(3):284-292). Polymerase chain reaction (PCR) has been used to identify class I alleles, but there is no published report of successful amplification of class III alleles.
More importantly, the conventional Southern blotting approach only allows classification of individuals as having no class III allele (genotype: I/I) or having at least one class III allele (genotype I/III or III/III). Individuals in the latter category will be assigned a risk that is approximately 4-fold less (Bennett S T et al., 1996, Ann. Rev. Genet. 30:343-370), and this estimate will be entered in a formula along with information from other IDDM loci, in order to calculate risk. The risk assignment can be erroneous in individuals carrying the specific alleles S1 or S2.
Given the foregoing, it would be desirable to develop a novel DNA assay for the diagnosis and/or prediction of autoimmune diabetes which overcomes the drawbacks of the prior art. More particularly, an assay by which alleles predisposing for diabetes could be distinguished from protective alleles would be desirable.
One aim of the present invention is to provide a PCR-based method to distinguish not only between class I and class III alleles of the insulin VNTR, but also between different alleles within class III.
Accordingly, in one aspect, the present invention provides a DNA assay for the prediction of autoimmune diabetes in a human subject where predisposition to autoimmune diabetes in a human subject is indicated by the presence of at least one Class III allele of the INS VNTR which silences thymic insulin expression, said assay comprising the steps of:
a) obtaining a DNA sample from the subject and subjecting the sample to PCR amplification using a primer pair specific for class III alleles of the INS VNTR;
b) subjecting the amplified sample to digestion with a restriction enzyme having a cleavage site on an uncommon variant of the repeat unit of the Class III allele; and
c) subjecting the digested sample to electrophoresis to identify the presence of a class III allele which silences thymic insulin expression.
Specifically, the method of the present invention can reliably distinguish alleles in class III that silence thymic insulin expression from those that enhance thymic insulin expression. This distinction is very important, as the present genetic studies show that such xe2x80x9csilencingxe2x80x9d class III alleles are predisposing to diabetes, while most class III alleles are protective. As used herein, the phrase xe2x80x9csilence thymic insulin expressionxe2x80x9d refers to the lack of insulin mRNA expression in the thymus of individuals possessing certain class III alleles.
The DNA assay of the present invention can be easily adapted to a miniaturized, automated genotyping approach utilizing fluorescent labeling. This genotyping will be an important part of a panel of genotypes that will determine diabetes risk.
The assay of the present invention is PCR-based, which makes it ideal for miniaturization and automation. The applicants are the first to develop a protocol for the successful amplification of class III insulin VNTR, technically a challenging task, as it involves amplification of highly repetitive GC-rich fragments of 2-3 kb length, well beyond the sizes handled by conventional PCR.
It distinguishes xe2x80x9csilencingxe2x80x9d class III alleles, such as the S1 and S2 alleles (described in greater detail herein), from other class III alleles, and assigns a higher rather than a lower risk to them, which will improve both the sensitivity and the specificity of the method. This is the main advantage of the assay of the present invention over the prior art.
To illustrate the importance of the assay of the present invention, the following must be considered:
The applicants have examined 167 diabetic children. Of these, 16 had a paternally transmitted S1 or S2 allele (high-risk class III). The conventional method, unable to distinguish S1 and S2 alleles, would have assigned to them the four-fold lower risk associated with class III as a whole. The assay of the present invention assigns them the true risk conferred by these alleles, which is 4.6-fold higher.
Thus, 16 of 167 children, almost 10%, would have been erroneously given a risk sixteen-fold lower than appropriate. Ten per cent of the population may not seem to be much, but it must be born in mind that in a complex disease like diabetes, a universal risk determination must be pieced together from evaluation of small effects at each locus. For this reason, the assay of the present invention is very likely to become an indispensable part of any attempt to predict diabetes.
In a preferred embodiment of the present invention, the DNA assay is employed to identify S1 and S2 class III alleles of the insulin VNTR whereby identification of an S1 or S2 allele is indicative of at least 1% risk of autoimmune diabetes, which is  greater than 10 fold higher than that erroneously predicted by existing methods of the prior art.
The INS VNTR is composed of a variable number of tandem 14-15 bp repeat sequences, with the consensus repeat unit ACA GGGG TGT GGGG (SEQ ID NO:1).
The amplification of step a) is effected using at least one primer pair selected from the group consisting of VNTR5 (TCAGGCTGGACCT CCAGGTGCCTGTTCTG) (SEQ ID NO:2)/VNTR6 (GCTGGTCCTGAGGM GAGGTGCTGACGA) (SEQ ID NO:3) and VNTR7 (GGCATCTTGGGCC ATCCGGGACTG) (SEQ ID NO:4)/VNTR8 (GCAGGGCGGGGCTCTTTGCGCTG) (SEQ ID NO:5).
The sample is selected from the group consisting of blood, saliva, urine and hair follicle.
The electrophoretic co-migration of step b) is effected using PAGE.
In another aspect of the present invention, isolated Class III alleles of the INS VNTR associated with silencing of thymic insulin mRNA expression are provided, including for example, S1 and S2 alleles.
In a further aspect of the present invention, primer pairs for PCR amplification of class III alleles of variable number of tandem repeats (VNTR) located upstream of the insulin gene (INS) are provided comprising: VNTR5 (TCAGGCTGGACCTCCAGGTGCCTGTTCTG) (SEQ ID NO:2)/VNTR6 (GCT GGTCCTGAGGAAGAGGTGCTGACGA) (SEQ ID NO:3) and VNTR7 (GGCATCT TGGGCCATCCGGGACTG) (SEQ ID NO:4) VNTR8 (GCAGGGCGGGGCTCTTT GCGCTG) (SEQ ID NO:5).